DESCRIPTION: The objective of the proposed work is to use optogenetics to investigate key signaling pathways - Wnt and Rho - in adult neural stem cells (NSCs) and embryonic stem cells (ESCs). Stem cells are being broadly explored for their potential for tissue regeneration, including strategies to manipulate endogenous stem cells residing within a patient's tissues (e.g., adult neural stem cells) as well as to implant exogenously cultured stem cells (e.g., embryonic or induced pluripotent stem cells) or their differentiated progeny. However, basic knowledge of the signaling mechanisms that control stem cell self-renewal and differentiation is currently incomplete, which limits our understanding of their role in adult function and complicates efforts to precisely control stem cell behavior for regenerative medicine applications. Light is a powerful tool to investigate cellular function. In particular, the abilityto modulate the Wnt and Rho signaling pathways spatially and temporally would allow us to elucidate their role in controlling stem cell fate, which is critical for biomedical applications. o this end, we recently developed optogenetic methods that enable a light input to be channeled into several defined signaling pathways through the assembly of nanoscale signaling complexes. Specifically, we engineered a photo-activatable agonist of the canonical Wnt signaling pathway as well as the RhoA pathway. We also have the ability to use microscopy techniques to conduct single molecule fluorescent imaging at nanoscale resolutions and to use biosensors based on fluorescence resonance energy transfer (FRET) to characterize Rho GTPase activities in live cells in real time. The first aim of the proposed work is to determine whether optogenetic activation of Wnt/?-catenin signaling in NSCs and ESCs can elucidate mechanisms of stem cell regulation. The second aim is to use optogenetics to probe Rho GTPase signaling and crosstalk in NSCs. The third aim is to determine whether Cry2 can be engineered for cell surface receptor activation and for multiplexed stimulation of cellular signaling. We anticipate that applying our novel optogenetic approaches will elucidate the dynamic roles of the Wnt and Rho pathways in regulating cell fate choices in NSCs and ESCs. Moreover, the novel optogenetic methods that we will develop will further enhance future investigations of key signaling pathways in cell and stem cell biology. The resulting mechanistic insights into stem cell biology and engineering will greatly impact approaches to restore organ function based on cell replacement and regenerative medicine.